Abstract
Serum free cultivation of Leishmania is cost-effective and improves large scale production of well defined parasite material. Moreover, the production of recombinant pharmaceutical proteins requires cultivation of the host in a culture medium free of animal materials, so several culture media for Leishmania tarentolae expression system have been introduced. Some investigations have established the development of a serum-free, but hemin containing medium, based on yeast extract and buffer salts. Hemin is a substance of animal origin also interferes with nickel ions on Ni-NTA resin. In this study, L. tarentolae from Iranian lizard, cultivated in a compound serum and hemin free medium (LBR medium) and the growth parameters were determined. Here we report that LBR medium could obtain high maximal cell density of 1.8 × 108 cells ml -1 equivalent to that of hemin containing medium in our conditions. This compound medium was confirmed by successful expression of a 28 kDa his-tagged protein. With knowledge of the results, the easy-preparing culture medium could be used as a new culture medium for the production of recombinant proteins in L. tarentolae . Keywords: Leishmania tarentolae , serum free cultivation of Leishmania , protein expression, Leishmania culture media.
Highlights
The protozoan parasites of the genus Leishmania have a two-stage life cycle (Peters and Killick-Kendrick, 1987)
Leishmania tarentolae is a parasite of the gecko Tarentolae annularis (Wallbanks et al, 1985) and has been introduced as a novel eukaryotic expression system for the production of recombinant proteins with mammalian-like post translational modification pattern (Fritsche et al, 2007)
Culture medium was supplemented with 10% of Fetal bovine serum (FBS) (Gibco, Germany)
Summary
The protozoan parasites of the genus Leishmania have a two-stage life cycle (Peters and Killick-Kendrick, 1987). BHI-FBS Brain Heart infusion + FBS et al, 2002) The advantages of this system are easy handling, the higher growth rate and cultivation in lower cost medium compared to mammalian cells (Phan et al, 2009). Promastigotes are mainly cultivated in liquid media to which animal serum, blood or hemin is added (Limoncu et al, 2004). A commonly used medium is Brain Heart Infusion (BHI), partially supplemented with serum or hemin (Cox and Hardegree, 1976; Fritsche et al, 2007). We tried to develop a nutrient medium free of animal substances for cultivation of L. tarentolae as a host for the production of recombinant proteins as therapeutic agents. The growth parameters of the wild type organism and an expressing type, such as the rate of cell division (r), time of doubling (t), and maximal cell density (Nmax) were determined
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